ABSTRACT
Objectives During the COVID-19 pandemic, several laboratories used different RNA extraction methods based on the resources available. Hence this study was done to compare the Ct values in qRT-PCR, time taken (sample processing-loading to PCR), manpower requirement, and cost of consumables between manual and automated methods. Materials and methods A cross-sectional study was done on 120 nasopharyngeal/oropharyngeal swabs received in VRDL for RT-PCR testing. Based on the results of automated RNA extraction (Genetix, HT 96 Purifier) and RT-PCR (Trivitron PCR Kit) detecting E gene (screening) and ORF gene (confirmatory), the division into Group- I (Ct 15-22), Group- II (Ct 23-29), Group-III (Ct 30-36) and Group-IV (Ct >36) was done. Manual RNA extraction was done using magnetic beads (Lab system, Trivitron). Statistical analysis Data were analyzed by SPSS 19.0 version software. Ct values obtained in the two methods were compared by paired t-test, GroupWise. Z test was used to compare the other parameters. Results The difference in Ct values for target genes was statistically significant (p<0.05) in Group-I to III; however, no variation in result interpretation. The difference in time, manpower, and cost were statistically significant (p<0.05). The manual method required twice more manpower; 40 minutes more time & automated method cost 3.5 times more for consumables. Conclusion The study showed that RNA yield was better with automated extraction in comparison to manual extraction. The samples extracted by the automated method detected the virus at a lower Ct range by PCR than the manual method. Automated method processed samples in less time and with less manpower. Considering the cost factor, manual extraction can be preferred in resource-limited settings as there was no difference in the results of the test. The manual method requires more hands-on time with potential chances of cross-contamination and technical errors.
ABSTRACT
Rapidly Growing Mycobacteria (RGM) are opportunistic pathogens found in the environment. Mycobacterium fortuitum, M. chelonae and M.abscessus are the important human pathogens of this group. They cause wound infections, disseminated cutaneous disease, pulmonary infection in patients with cystic fibrosis or bronchiectasis, bone and joint infections and keratitis. Infections due to these Non-Tuberculous Mycobacteria (NTM) are increasingly reported. Post laparoscopic wound infections, mesh site infections and other surgical site infections due to M. fortuitum and M. chelonae have been reported. Usually wound infections due to atypical mycobacteria have delayed onset and do not respond to conventional antibiotics. Identification of RGM can be done by a set of cumbersome biochemical tests, High Performance Liquid Chromatography (HPLC), molecular methods using DNA probes or by Polymerase Chain Reaction (PCR). We here report a case of post-herniorrhaphy wound infection due to M. fortuitum which was identified by molecular method (HAIN mycobacterial species system). This case report underscores the importance of examining Ziehl-Neelsen (ZN) stain of all exudates with sterile culture on day one for non fastidious bacteria. Timely identification can lead to prompt therapy of patients preventing further complications.
